Abstract Glaucoma can be defined as progressive optic neuropathy with structural damage of the optic nerve (ON) head and selective death of retinal ganglion cells (RGC). In our previous studies we described glaucomatous alterations in the ON of glaucoma patients. We have found that g-synuclein is implicated in morphological changes in the ON and RGC that occur in glaucoma. In the search of the mechanism of 3- synuclein involvement in glaucomatous changes we found that g-synuclein is a modulator of cell death/cell survival. This function is realized by modulating the level of phosphorylation of proapoptotic member of Bcl-2 family, Bad. In addition, g-synuclein is a marker of RGC and a transcriptional regulator. One of the targets of g- synuclein is the matrix metalloproteinase-9 (MMP-9) gene. MMP-9 modulates extracellular matrix (ECM) and the alteration of ECM is a key step in glaucomatous damage in the ON. In this application we propose to investigate the role of g-synuclein in RGC, mechanisms of MMP-9 regulation and elucidate their implications for eye pathology Hypotheses: 1. Two important mechanisms regulating MMP-9 expression are: a). Upregulation of MMP-9 expression by g- synuclein at the transcriptional level and b). Post-transcriptional mechanisms including modulation of its translation/or mRNA stability by 3'-UTR elements. Defects in the regulation of MMP-9 lead to a misbalance of its expression, defects in the remodeling of the ECM and ultimately to eye pathology. 2. In addition to being transcriptional regulator of MMP-9, g-synuclein has other functions in RGC. g-Synuclein affects cell viability, electrical responses of RGC, modulates cell survival/cell death cascades and is implicated in glaucomatous alterations in the retina and ON. To test these hypotheses we propose the following specific aims: Specific aims: 1). Using animal and cellular models, examine g-synuclein functional role in normal retina and in glaucoma pathogenesis: A. g-Synuclein effect on RGC physiology (electroretinogram, ERG), MMP-9 (IHC and activity) and RGC viability (primary culture) will be studied by comparing these functions in g-synuclein knockout (KO) mice and wt animals. B. g-Synuclein effect on glaucoma pathogenesis will be investigated using mice generated as backcross of g- synuclein KO and DBA/2J glaucoma model mice. Retina and ON will be assessed in the offspring after backcross. C. The effect of partial reduction of 3-synuclein level on RGC will be assessed using RGC-5 culture. g- Synuclein level will be reduced by siRNA and cell viability, MMP-9 level and the effect on the expression of other genes by gene array will be assessed. 2). Reveal the molecular mechanisms responsible for the regulation of MMP-9 transcription. Develop efficient approaches to downregulate MMP-9 (siRNA technology, g-synuclein level reduction) and inhibit its activity (expression of MMP-9 inhibitor, TIMP-1). 3). Elucidate the molecular and cellular mechanisms that control g-synuclein transcription. Identify cis-elements in g-synuclein promoter that directs its activity. Investigate g-synuclein as a new marker of RGC and modulator of cell survival/cell death pathways. Studies proposed in this application will explore new mechanisms of glaucoma pathogenesis. Since defects in MMP-9 control play an important role in glaucoma and some retinal diseases, the results received will provide essential insight into understanding the mechanisms and developing treatments for these ocular pathologies. The experiments with g-synuclein will demonstrate the role of this protein in normal RGC and its involvement in glaucoma. Experiments described in Specific Aim 2will be a base for translational studies aimed at the treatment of glaucoma by gene therapy. PUBLIC HEALTH RELEVANCE: Glaucoma is the second leading cause of blindness worldwide. Approximately 4 million Americans have glaucoma and 120,000 are blind because of glaucoma. There is sound medical-scientific evidence that indicates that open-angle glaucoma can be related to relevant service rendered by veterans, members of Peacekeeping Forces, or members of the Armed Forces. Recent studies showed an important role of 3-synuclein as a modulator of retinal ganglion cell survival/death and regulator of extracellular matrix degrading enzymes (MMPs) and especially MMP-9 in eye cells. The purpose of this proposal is to examine how 3-synuclein affects prosurvival/prodeath signaling and how it is implicated in MMP-9 regulation in glaucoma.